The Effect of Oxytocin and an Oxytocin Antagonist on the Human Myometrial Proteome
In: Reproductive sciences: RS : the official journal of the Society for Reproductive Investigation, Band 17, Heft 1, S. 40-46
ISSN: 1933-7205
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In: Reproductive sciences: RS : the official journal of the Society for Reproductive Investigation, Band 17, Heft 1, S. 40-46
ISSN: 1933-7205
Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets. © 2012 Smith et al. ; Spanish MICINN (Granted projects BFU 2008-01566/BMC y and CSD2008-00013). EDN acknowledges support from the PRIME-XS project, grant agreement number 262067, funded by the European Union 7th Framework Programme and from the Basque Country Government. ; Peer Reviewed
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Modern omics technologies allow us to obtain global information on different types of biological networks. However, integrating these different types of analyses into a coherent framework for a comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics, and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRASmediated signaling is the strongest from the cell membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal distinct HRAS functions including the control of cell migration from the endoplasmic reticulum and TP53-dependent cell survival when signaling from the Golgi apparatus. ; This work was supported by the Science Foundation Ireland grants 06/CE/B1129, 14/IA/2395, and 15_CDA_3495; Irish Cancer Society BREAST-PREDICT grant CCRC13GAL; and by the European Union FP7 grant 278568 "PRIME-XS." T.-T.A. and A.J.R.H. were supported by the Roadmap Initiative Proteins@Work (184.032.201) funded by the Netherlands Organisation for Scientific Research (NWO). P.C. was supported by grant SAF-2015 63638R (MINECO/FEDER, UE); by Centro de Investigación Biomédica en Red de Cáncer; and by Asociación Española Contra el Cáncer (AECC), grant GCB141423113.
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Wnt/β-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multi-meric Wnt–FZD assemblies via intramembrane interactions. Subsequently, these Wnt–FZD–TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions. ; This work is part of the Oncode Institute which is partly financed by the Dutch Cancer Society. This work was supported by European Research Council Starting Grant 242958 (to M.M.M.), European Union Grant FP7 Marie Curie ITN 608180 "WntsApp" (to M.M.M.), and the Netherlands Organization for Scientific Research NWO VICI Grant 91815604 and ECHO Grant 711.013.012 (to M.M.M.). T.Y.L. and A.J.R.H. are supported by large-scale proteomics facility Proteins@Work Project 184.032.201 embedded in the Netherlands Proteomics Centre and supported by the Netherlands Organization for Scientific Research. They were additionally supported through European Union Horizon 2020 Program FET-OPEN Project MSmed, Project 686547. F.X.P. was supported by Grants SAF2014-53320R and SAF2017-88390R from the Spanish Government. ; Peer Reviewed
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Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. ; We thank Kento Onishi for providing EpiSC samples. We also thank Dr Jeff Wrana and Dr Dan Trcka for providing secondary DOX-inducible reprogramming system to allow further validation of our findings. We would also like to acknowledge the assistance and support of laboratory colleagues and collaborators. This work has been supported by a GL2 grant from Ontario Government (AN) and a CIHR Grant to P.W.Z. N.S. is the recipient of the NSERC Vanier Canada Graduate Scholarship. A.N. is Tier 1 Canada Research Chair in Stem Cells and Regeneration. P.W.Z. is the Canada Research Chair in Stem Cell Bioengineering. ; Sí
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Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC. ; We are grateful to the Spanish Biobanks integrated within the Spanish Network of National Biobanks for the tumor samples used in our investigations and to the Breast Cancer Group at VHIO for providing study materials. The present study was funded by FIS PI10/00288, FIS PI13/00430, and AECC Scientific Foundation "Beca de Retorno 2010"awarded to MQF; FIS PI11-00832, FIS PI14-00726, and ISCIII PIE15/00068 awarded to RC. I.Z. is a recipient of a La Caixa PhD Fellowship, 2011. S.F. is a recipient of a FPI grant (SEV-2015-0510-16-6). V.S. is supported by the Miguel Servet Program (CP14/00228). This work was also supported by the PRIME-XS project, Grant Agree- ment Number 262067, funded by the European Union Seventh Framework Program and the project Proteins At Work (project 184.032.201), a program of the Netherlands Proteomics Centre financed by the Netherlands Organisation for Scientific Research (NWO). ...
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Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
BASE
Glycosylation is a topic of intense current interest in the development of biopharmaceuticals since it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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